Ionotropic glutamate receptors (iGluRs) are membrane proteins that act as molecular pores and mediate signal transmission at the majority of excitatory synapses in the mammalian nervous system. iGluRs are also found in primitive prokaryotes and eukaryotes suggesting an early evolutionary origin, followed by gene expansion to generate subtypes with diverse functional properties. The 7 gene families of ionotropic glutamate receptors (iGluRs) in humans encode 18 subunits which assemble to form 3 major functional families named after the ligands which were first used to identify iGluR subtypes in the late 1970s: AMPA, kainate and NMDA. Because of their essential role in normal brain function and development, and increasing evidence that dysfunction of iGluR activity mediates multiple neurological and psychiatric diseases, as well as damage during stroke, a substantial effort in the Laboratory of Cellular and Molecular Neurophysiology is directed towards analysis of iGluR function at the molecular level. Atomic resolution structures solved by protein crystallization and X-ray diffraction provide a framework in which to design biochemical and electrophysiological experiments to define the mechanisms underlying ligand recognition, the gating of ion channel activity, and the action of allosteric modulators. This important information will allow the development of novel therapeutic reagents and reveal the inner workings of a complicated protein machine which plays a key role in brain function. CRYO EM STRUCTURES OF FULL LENGTH iGLURS Insight into molecular mechanisms underlying glutamate receptor gating is limited by lack of structural information for receptors trapped in different conformational states. We completed a structural analysis of an iGluR gating cycle, progressing from resting, to activated and then desensitized states. Comparison of the closed and active state electron density maps reveals LBD clamshell closure that produces a 7 vertical contraction of the ATD-LBD assembly, measured as a downwards movement at the top of the ATD tetramer, as well as unanticipated movements in the LBD, in which the dimer pairs rotate about an axis offset from the local axis of 2-fold symmetry. The net result of these movements is a novel corkscrew-like rotation that drives the transition from the closed to the active conformation. Analysis of cryo-electron microscopic images for the GluA desensitized state revealed evidence of substantial conformational heterogeneity precluding determination of a single desensitized state 3D structure. Three-dimensional classification enabled separation of three dominant classes, with variable degrees of displacement between ATD dimers compared to the closed and active states, at contrast to the rigid assembly of the ATD in GluK2. A structure of the GluK2 desensitized state, determined at 7.6 resolution, revealed electron density for all &#945;-helices in the ATD and LBD assemblies, and also for the M3 helix bundle, the upper segment of M1 and the pre-M1 cuff helix in the ion channel. The density map revealed preservation of 2-fold symmetry in the ATD layer while the LBD layer adopts a quasi 4-fold symmetric arrangement. The resolution of our map unambiguously shows that in the desensitized state the ion channel adopts a closed conformation in which the M3 helices form a crossed bundle assembly with the pre-M1 helices wrapped around the outside of the channel. The 4-fold symmetry in the LBD layer matches that of the ion channel in its non-conducting state, and thereby permits the channel to adopt a low energy conformation. It is notable that while both AMPA and kainate receptors adopt 4-fold symmetry in their desensitized LBD layers, in the AMPA receptor desensitization also causes a rupture in the ATD layer. This result can be understood by considering symmetry mismatch within the receptor, and changes in symmetry during the gating cycle. In the closed and open states, both the ATD and LBD layers have 2-fold symmetry. The strain resulting from agonist binding to the LBD is centered near the LBD-TM interface and is sufficient to open the channel. In the desensitization step, the LBD layer shifts from 2-fold to 4-fold symmetry, matching the 4-fold symmetry of the ion channel; the strain in the receptor now shifts to the 2-fold symmetric ATD. In GluK2 the ATD assembly appears to be able to withstand this strain, possibly relieving it by a drawbridge-like tilting at the ATD tetramer interface. However, in GluA2, this symmetry mismatch places sufficient strain on the ATD layer to rupture the tetramer interface. FUNCTIONAL RECONSTITUTION OF DROSOPHILA NMJ GLUTAMATE RECEPTORS The Drosophila larval neuromuscular junction, at which glutamate acts as the excitatory neurotransmitter, is a widely used model for genetic analysis of synapse function and development. Despite decades of study, the inability to reconstitute neuromuscular glutamate receptor function using heterologous expression systems has complicated the analysis of receptor function, such that it is difficult to resolve the molecular basis for compound phenotypes observed in mutant flies. We performed electrophysiological studies to test if the auxiliary subunit Neto was required for the functional reconstitution of Drosophila NMJ iGluRs. This revealed that the major effect of Neto was to increase receptor activity, with only a small effect on receptor trafficking. We established that four different iGluR subunits are required for robust expression; that Drosophila NMJ iGluRs are Ca2+ permeable and exhibit voltage dependent channel block by cytoplasmic polyamines; and that they have a ligand binding profile different from that of vertebrate AMPA, kainate and NMDA receptors. To investigate the structural basis for this unique profile we identified GluRIIB as a promising candidate for crystallization. X-ray diffraction data for the GluRIIB S1S2 complex with glutamate, at a resolution of 2 , revealed the classical back to back LBD dimer assembly, as first reported for the GluA2 AMPA receptor with glutamate bound in a cavity of volume 208 3 together with three trapped water molecules. Within the binding site, the side chain of Asp509 forms a hydrogen bond with the hydroxyl group of Tyr481, a conserved aromatic residue that caps the entrance to the ligand binding cavity; this interaction locks these residues in place, producing steric clashes that prevents binding of AMPA and kainate. Amino acid sequence alignments reveal that Asp509 is conserved in all Drosophila NMJ iGluRs, while in all vertebrate AMPA and kainate receptor subunits there is a proline at this position. CTENOPHORE GLUTAMATE RECEPTORS Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, while ligand-binding assays show that glycine binds with nM affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species.